We have developed a new method for estimating the localization of DNA damage such as apurinic/apyrimidinic sites (APs) on DNA using fluorescence anisotropy. This method is aimed at characterizing clustered DNA damage produced by DNA-damaging agents such as ionizing radiation and genotoxic chemicals. A fluorescent probe with an aminooxy group (AlexaFluor488) was used to label APs. We prepared a pUC19 plasmid with APs by heating under acidic conditions as a model for damaged DNA, and subsequently labeled the APs. We found that the observed fluorescence anisotropy (robs) decreases as averaged AP density (AP: number of APs per base pair) increases due to homo-FRET, and that the APs were randomly distributed. We applied this method to three DNA-damaging agents, 60Co -rays, methyl methanesulfonate (MMS), and neocarzinostatin (NCS). We found that robs–AP relationships differed significantly between MMS and NCS. At low AP density (AP < 0.001), the APs induced by MMS seemed to not be closely distributed, whereas those induced by NCS were remarkably clustered. In contrast, the AP clustering induced by 60Co -rays was similar to, but potentially more likely to occur than, random distribution. This simple method can be used to estimate mutagenicity of ionizing radiation and genotoxic chemicals.